Creatine and creatinine are usually found in blood and urine, and can be used as very important parameters for diagnosing uremia, chronic nephritis, acute nephritis, gigantism, tonic muscular dystrophy and some other related diseases. For such routine diagnostic purposes, creatinine and creatine concentrations in blood and urine need to be determined quickly and accurately (Schumacher, G., et al., Ann Biol Clin 51 (1993) 815-9, Fujita, T., et al., Clin Chem 39 (1993) 2130-6, Spencer, K., Ann Clin Biochem 23 (1986) 1-25).
Generally, creatinase catalyses the hydrolysis of creatine (e.g. as generated from creatinine through creatinase) to urea and sarcosine. In the presence of sarcosine oxidase, sarcosine is oxidized and hydrogen peroxide is generated. The hydrogen peroxide formed is determined by a standard procedure for measurement of hydrogen peroxide, e.g. by a colorimetric procedure catalyzed by horse-radish peroxidase. Through these coupled reactions, creatinine and/or creatine in a sample can be quickly and quantitatively determined (Kopetzki, E., et al., Clin Chem 40 (1994) 688-704, Beyer, C., et al., Clin Chem 39 (1993) 174-34).
Enzymes with creatinase activity have been found in different microorganisms, including Arthrobacter (Kaplan, A. Szabo, L. L., Mol Cell Biochem 3 (1974) 17-25, Kaplan, A. Naugler, D., Mol Cell Biochem 3 (1974) 9-15, Nishiya, Y., et al., Mol Gen Genet 257 (1998) 581-6), Bacillus (Suzuki, K., et al., Journal of Fermentation & Bioengineering 76 (1993) 77-81), Alcaligenes (Matsuda, Y., et al., Chem. Pharm. Bull. 34 (1986) 2155-2160), Pseudomonas (Hong, M. C., et al., Biochem Genet 36 (1998) 407-15) and Flavobacterium (Koyama, Y., et al., Agric Biol Chem 54 (1990) 1453-7).
Several creatinase enzymes obtained from different microorganisms have been industrially produced and used as reagents for clinical tests. However, even with state-of-the-art creatinase enzymes and/or with state-of-the-art creatine assays, the creatinase enzyme is generally the major limiting factor for or in such assays.
Important criteria for broad industrial, especially diagnostic applicability, comprise solubility, conductivity (in case of electronic signal generation and measurement), stability, and enzymatic activity with creatine as substrate. Obviously, stability (e.g., short-term heat stability or long-term storage stability) and enzymatic activity are especially relevant or critical in routine applications. Schumann et al. (Schumann, J., et al., Biol Chem Hoppe Seyler 374 (1993) 427-344) showed that “extrinsic factors”, such as DTE, bovine serum albumin (BSA) and glycerol, can improve the stability of P. putida creatinase. But these extrinsic factors are generally not compatible with the conditions for a coupled creatinine and/or creatine assay system using creatinase, creatinase, sarcosine oxidase and peroxidase.
All the native or wild-type creatinase enzymes known from the art exhibit certain limitations with regard to thermostability and their Km value for creatine. For example, the enzyme derived form the Bacillus is unstable at any temperature of more than 40° C. (see patent U.S. Pat. No. 4,420,562). The creatinase from Pseudomonas putida has both, rather low storage stability (Schumann, J., et al., supra) and a rather high Km value (see patent EP 0 291 055), and, consequently, the enzyme is not suitable for creatine determination in a liquid formulation as required in the coupled creatine assay mentioned above using also sarcosine oxidase and peroxidase as auxiliary reagents.
Creatinases isolated from Corynebacterium (see patent DE 2 659 878), Micrococcus, or Bacillus (see patent DE 3 024 915) have been found unstable at elevated temperatures and, therefore are not suitable for the formulation of long-term stable liquid assay reagents, either. A creatinase from Alcalingenes has been reported to have a Km value of 13 mM and to be stable at a temperature of 45° C. for 30 minutes (see patent U.S. Pat. No. 5,451,520).
There are few literature reports about engineered creatinases (Schumann, J., et al., Protein Sci 2 (1993) 1612-20) used random and site-directed mutagenesis to introduce point mutations into P. putida creatinase in order to generate mutants with an altered stability profile. Three point mutants with A109V, V355M, or V182I, one double mutant with A109V and V355M, and one triple mutant with all three substitutions in P. putida creatinase were generated and compared to the wild-type enzyme regarding their physical and enzymological properties. Physicochemical measurements showed that the mutations exhibit only a small increase in overall stability. Even the best mutant, however, is not stable at elevated temperature, e.g. temperatures of 45° C. and above. The catalytic properties are in the range of the wild-type enzyme or are inferior.
EP 0 790 303 describes a process of generating Alcaligenes faecalis crcatinase variants with lower Km values as compared to the wild-type enzyme using the in vivo mutagenesis approach in E. coli strain XLl-Red (Stratagene, CaL Nr. 200 129). The Km values of the creatinase variants were found to range from 4.5 mM to 9.0 mM.
It can be summarized that the attempts known from the art, e.g., as discussed above, either aiming at the isolation and purification of creatinase from various microorganisms or at the genetic improvement of such enzymes, to date still have led to enzymes lacking important industrial properties.
A great demand and clinical need therefore exists for mutant forms of creatinase with improved properties, especially regarding improved stability and/or lower Km-value to its substrate creatine.
It was the task of the present invention to provide new mutants or variants of creatinase with significantly lower Km-value or significantly improved stability, or both.
Surprisingly it has been found that it is possible to provide such creatinase enzymes by mutating one or several amino acids of an Erwinia-type creatinase at precisely defined positions. Such relevant positions are defined and given as amino acid positions corresponding to amino acid positions of the wild-type creatinase isolated from Erwinia sp. DSM 97-934.